Type 1 allergies are of worldwide importance. Up to 25% of the population of industrialised countries suffer from complaints such as allergic rhinitis, conjunctivitis or bronchial asthma which are caused by allergens of various origin present in the air (aeroallergens), such as plant pollen, mites, cats or dogs. Up to 40% of these type 1 allergy sufferers in turn exhibit specific IgE (immunoglobulin E) reactivity in the case of grass pollen (Freidhoff et al., 1986, J. Allergy. Clin. Immunol. 78, 1190-201).
The substances which initiate type 1 allergy are proteins, glycoproteins or polypeptides. After uptake via the mucous membranes, these allergens react with the IgE molecules bonded to the surface of mast cells in sensitised people. If two IgE molecules are crosslinked to one another by an allergen, this results in the release of mediators (for example histamine, prostaglandins) and cytokines by the effector cell and thus in the corresponding clinical symptoms.
Depending on the relative frequency of the allergy sufferers having IgE antibodies against certain allergens, a distinction is made between major and minor allergens. In the case of timothy grass (Phleum pratense), Phl p 1 (Petersen et al., 1993, J. Allergy Clin. Immunol. 92, 789-796), Phl p 5 (Matthiesen and Löwenstein, 1991, Clin. Exp. Allergy 21, 297-307), Phl p 6 (Petersen et al., 1995, Int. Arch. Allergy Immunol. 108, 49-54) and Phl p 2/3 (Dolecek et al., 1993) have hitherto been characterised as major allergens and Phl p 4 (Löwenstein, 1978, Prog. Allergy 25, 1-62) and group 10 and 11 from Lolium perenne (Ansari et. al., 1987, J. Allergy Clin. Immunol. 80, 229-235) as minor allergens.
Group 1, which includes Phi p 1 from timothy grass, is classified as one of the most relevant allergen groups of grass pollen (Tamborini, E. et al., Eur. J. Biochem. 1997, 249:886-894). The other representatives of group 1 from other grasses have homologies of in some cases more than 95% to Phl p 1 (Petersen, A., et al., J. Allergy Clin. Immunol. 1995, 95: 987-994). Owing to the high homologies, reactions to the allergens of other cross-reactive species also occur in the case of sensitisation with a grass. For this reason, these molecules are of overriding importance for corresponding diagnostic and therapeutic approaches.
On therapeutic use of these allergens, use is made of the reaction with T helper cells, where reorientation of the pathological TH2 cells into the TH1 type occurs. This causes a change in the cytokine profile so that B cells are stimulated to form IgG instead of IgE.
A classical approach to effective therapeutic treatment of allergies is specific immunotherapy or hyposensitisation (Fiebig, 1995, Allergo J. 4 (6), 336-339, Bousquet et al., 1998, J. Allergy Clin Immunol. 102(4), 558-562), in which the patient is injected subcutaneously with natural allergen extracts in increasing doses.
However, there is a risk in this method of allergic reactions or even anaphylactic shock. In order to minimise these risks, innovative preparations in the form of allergoids are being employed. These are chemically modified allergen extracts which have significantly reduced IgE reactivity, but identical T-cell reactivity compared with the untreated extract (Fiebig, 1995, Allergo J. 4 (7), 377-382).
Even more substantial therapy optimisation would be possible with allergens prepared by recombinant methods. Defined cocktails, optionally matched to individual patients, of highly pure allergens prepared by recombinant methods could release extracts from natural allergen sources since these, in addition to the various allergens, contain a relatively large number of immunogenic, but non-allergenic accompanying proteins. Realistic perspectives which may result in reliable hyposensitisation with expression products are offered by specifically mutated recombinant allergens in which IgE epitopes are specifically deleted without impairing the T-cell epitopes which are essential for therapy (Schramm et al., 1999, J. Immunol. 162, 2406-2414).
A further possibility for therapeutic influencing of the disturbed Th-cell equilibrium in allergy sufferers is treatment with expressible DNA which encodes for the relevant allergens. Initial experimental evidence of allergen-specific influencing of the immune response has been furnished in rodents by ejection of allergen-encoding DNA (Hsu et al., 1996, Nature Medicine 2 (5), 540-544).
Phl p 1 is a protein comprising 240 amino acids and an n-glycosylation site. The glycosylation fraction is 5% of the molecular weight, which, in the natural protein, is about 30-35 kDa (Petersen et al., Allergy Clin. Immunol. 1995, 95: 987-994; Suck et al., J. Immunol. Meth. 1999, 229:73-80). The nucleic acid sequence of Phl p 1 is known (Laffer et al., J. Allergy Clin Immunol. 1994, 94: 689-698; Petersen et. al., J. Allergy Clin. Immunol., 1995, 95: 987-94) and can thus be utilised for the recombinant preparation of the molecule.
Previous attempts to prepare the molecule by recombinant methods in bacterial or eukaryotic systems, such as, for example, yeast, in such a way that a stable monomeric form was obtained were, however, unsuccessful owing to its poor solubility:
In the case of bacterial expression, Phl p 1 is deposited as inclusion bodies (Vrtala et al., J. Allergy Clin. Immunol, 1996; 97: 781-7) and firstly has to be denatured before purification. The denaturing agent is subsesequently removed. However, complete re-folding of the protein into the natural soluble conformation has hitherto not been achieved (Andersson, Lidholm, Int. Arch. Allergy Immunol. 2003; 130:87-107).
One possible reason preventing the formation of a stable conformation could have been the absence of glycosylation. However, a stable Phl p 1 has not been obtained even in eukaryotic systems in which glycosylation is possible (K. Grobe, Dissertation, 1998, University of Hamburg).
Instead, a cause of the lack of solubility is assumed to be proteolytic activity, which results in self-degradation of the molecule (Grobe et al., Eur. J. Biochem. 1999; 263: 3340; Kirsten Gehlhar, Dissertation, 1998, Medical University of Lübeck, Germany). In addition, hydrophobic interactions between the molecules are also possible as a cause of the aggregation.
The object on which the present invention is based thus consisted in the provision of variants of the major allergen Phl p 1 from timothy grass which are distinguished by improved solubility at the same time as full retention of the therapeutically and diagnostically important immunological properties and which can thus be purified in pharmaceutically suitable form.